The regulation of semiconservative and repair-type DNA synthesis in normal and transformed human cells was compared by ascertaining the following: 1) the effects of the disruption of higher-order DNA-protein structure upon DNA synthesis; 2) the utilization of the thymidine (TdR) analogue 5-bromodeoxyuridine (BUdR) by the DNA replication apparatus; and 3) the effects of specific inhibitors of mammalian DNA polymerases upon DNA synthesis in situ. Novobiocin - an inhibitor of the bacterial enzyme DNA gyrase which catalyzes DNA supercoiling - blocks semiconservative and repair DNA replication in all cells tested, as well as the replication in situ of adenovirus DNA. Treatment of human (KD) and Chinese hamster ovary (CHO) cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and/or novobiocin produced surviving cells whose sensitivity to the inhibition of DNA synthesis by novobiocin is less than that of untreated cells. BUdR was incorporated into the DNA of diploid human fibroblasts far less efficiently than into that of chemically-transformed KD cells or of cells derived from human brain tumors. DNA synthesis in some transformed cells was less sensitive to N-ethyl-maleimide (NEM) - a selective inhibitor of mammalian DNA polymerase alpha.